Human and simian immunodeficiency viruses encode a number of accessory proteins that have been highly conserved throughout viral evolution yet are not absolutely required for viral replication in vitro. Studies of the functions of these accessory proteins are important because of their demonstrated importance in vivo and the possibility that their genetic manipulation may provide important new approaches to HIV vaccine and drug development. Two proteins, vpx and vpr, are of particular interest because they are packaged into virus particles in quantities comparable to structural proteins and are required for full viral infectivity in natural (primary) target cells. During the last funding period, we showed that the vpx open reading frame encodes a 14-16 kDa protein which is expressed in vivo, that vpx is incorporated into virions in amounts comparable to gag, that vpx is required for wild-type levels of viral replication in primary (but not immortalized) cells, and that vpx effects are seen early in the viral life cycle at the level of cell entry and/or reverse transcription. We also demonstrated that vpx is packagable into wild-type and vpx-deficient virions when supplied in trans, that virion incorporation requires a specific interaction with the gag polyprotein, and that vpx fusion proteins can be constructed that mediate the packaging of "foreign" proteins into the virus particle. Finally, we discovered a novel 30 kDa vpx-immunoreactive protein in HIV-2 and SIV virions which we believe represents a "natural" fusion protein generated by alternative splicing of vpx to another viral gene, targeting the later product to the virion. In this renewal application, we propose to continue to study the biosynthesis of vpx and vpr and elucidate their mechanisms of action. We will employ the SIV(sm)PBj virus system so that the function of native and genetically-altered vpx and vpr proteins can be assessed in vitro and in vivo. The specific aim will be to characterize the replication patterns, natural history and pathogenicity of wild-type versus vpr, vpx, and vpr/vpx deficient SlV(sm)PBj viruses in the pig-tailed macaque animal model system. This is expected to yield new insights into the mechanism of action of vpx and vpr, and to provide basic knowledge for novel strategies of rational drug design and vaccine development against HIV/SIV.